Inhibition of Penicillium digitatum and Penicillium italicum in vitro and ...

Antonie van Leeuwenhoek (2011) 99:85–91 DOI 10.1007/s10482-010-9527-0

ORIGINAL PAPER

Inhibition of Penicillium digitatum and Penicillium italicum in vitro and in planta with Panomycocin, a novel exo-b-1,3-glucanase isolated from Pichia anomala NCYC 434 Demet Altınbay Izgu • Remziye Aysun Kepekci Fatih Izgu

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Received: 30 June 2010 / Accepted: 29 October 2010 / Published online: 14 November 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Panomycocin, a novel exo-beta 1,3 glucanase, was tested as an antifungal agent against green and blue mold diseases, the most important causes of post harvest decay in citrus fruits. All tested isolates of Penicillium digitatum and Penicillium italicum were susceptible to panomycocin in vitro. Effective panomycocin concentrations for 50% growth inhibition (MIC-2) for P. digitatum and P. italicum were 2 and 1 lg ml-1, respectively. Complete (MIC-0) growth inhibition of all isolates observed at a panomycocin concentration of 16 lg ml-1. Treatment of spores with panomycocin at values lower than the MIC-0 led to slower germ tube elongation and mycelium growth. In tests on fruit, panomycocin at concentrations equal to in vitro MIC-0 value protected lemon fruit from decay. Keywords Antifungal protein Exo-b-1,3-glucanase Panomycocin Citrus fruit Postharvest decay

D. A. Izgu Department of Biology, TED Ankara College Foundation High School, Incek, Go¨lbas¸ ı, 06830 Ankara, Turkey R. A. Kepekci F. Izgu (&) Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey e-mail: [email protected]

Introduction Fruit crops are particularly susceptible to fungal infections in the post harvest period due to their low pH, water content, nutrient composition, and loss of natural resistance while attached to the tree (Droby et al. 1992). Citrus is the world’s premier fruit crop grown commercially in more than 100 countries across six continents (Terol et al. 2007). They contain several phytochemicals and/or nutraceuticals including vitamin C with disease-preventing and lifesustaining functions (Dillard and German 2000). The commercial life span of harvested citrus is frequently reduced or terminated by fungal pathogens causing fruit decay (Liu et al. 2007). Mold growth in citrus fruits can also lead the production of allergenic spores and hazardous mycotoxins (Moss 2008). Currently, control of post harvest fungal diseases of citrus fruits relies mainly on the use of synthetic fungicides (Lo´pez-Garcia et al. 2000). However, due to the biologically dynamic nature of fungal population resistance has been encountered and this limits the effectiveness of those fungicides (Chen et al. 2008; Makovitzki et al. 2007). Moreover, growing public health and environmental concerns have resulted in the de-registration of some of the more effective synthetic fungicides (Makovitzki et al. 2007). Thus, there is a growing interest in the development of safer and effective alternative compounds to control post harvest fungal diseases. Among the different approaches, the discovery of

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naturally occurring antifungal proteins with little or no environmental impact and mammalian toxicity and a low tendency to elicit resistance are attracting increasing attention (Bobek and Situ 2003). Antifungal proteins are naturally produced by a diverse group of organisms including bacteria, fungi, insects, vertebrates and invertebrates as well as plants (Selitrennikoff 2001). Within those proteins, the yeast killer proteins are highly suggested as potential antifungal agents. Certain yeast strains with a killer phenotype (K?) produce extracellular protein toxins designated as killer proteins or killer toxins which are lethal to sensitive microbial cells (Hemandez et al. 2008; Schmitt and Breinig 2002). Killer protein production appears to be a widespread characteristic among yeast species of different genera including Saccharomyces, Hansenula, Kluyveromyces, Pichia and several others and this confers to yeast cells an ecological advantage over their competitors (Comitini et al. 2009; Magliani et al. 2008). There is considerable amount of published information on the wide range intergeneric killing spectrum of Pichia toxins (Sangorrin et al. 2001). Among the species with a killer phenotype, Pichia anomala NCYC 434 has been studied extensively and its killer protein, Panomycocin, has been suggested as a potential antifungal agent (Izgu and Altinbay 2004). Panomycocin is a glycosylated monomeric protein with a molecular mass of 49 kDa and belongs to the exo b1,3 glucanase group. It is highly stable at pHs between 3 and 5.5 and temperatures up to 37°C. Panomycocin exerts its cytocidal activity by hydrolyzing the b-1,3-glucans which are the primary polymers within the fungal cell wall (Izgu et al. 2005). Hydrolysis of this polymer leads to leakage of cytoplasmic components and ultimately cell death. Exo b-1,3 glucanases have potential applications in food, feed, agricultural, and fermentation industries (Ferrer 2006). Recently, we have reported the in vitro antifungal activity of Panomycocin against human dermatophytes (Izgu et al. 2007a) and Candida spp. (Izgu et al. 2007b). In the present study, we determined the in vitro and in vivo activities of this exo b-1,3 glucanase against isolates of P. digitatum and P. italicum, the two most common post harvest fungal decay organisms which cause green mold and blue mold diseases, respectively, in citrus fruits (Wuryatmo et al. 2003).

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Antonie van Leeuwenhoek (2011) 99:85–91

Materials and methods Fungal strains The Panomycocin producing strain, Pichia anomala (NCYC 434), was purchased from the National Collection of Yeast Cultures, Norwich, UK and maintained on YEPD agar (BactoTM yeast extract [Bectone, Dickinson Co., Sparks, Md., USA] 1% (w/ v), BactoTM peptone 2% (w/v), dextrose 2% (w/v), and BactoTM agar 2% (w/v), pH 5.5). Standard phytopathogenic fungi strains were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and included P. digitatum DSMZ 2776 and P. italicum DSMZ 2756. Field isolates of P. digitatum (PHD-7 and PHD-9) and P. italicum (PHI-2 and PHI-15) were obtained from diseased citrus fruits in Antalya, Turkey with typical green and blue mold symptoms, respectively. Isolations were made by removing the fruit skin from the margin of a lesion and asceptically placing bits of decayed tissue on petri plates containing *12 ml of potato-dextrose agar (PDA) and incubated at 20°C for 5–14 days. Strains were identified by the key system of Pitt (2000) using standard parameters such as morphology and growth on three standard media: Czapek-yeast agar (CYA), malt extract agar (MEA) and 25% (v/v) glycerol nitrate agar (G25 N). The most aggressive isolates which produced the largest lesions on inoculated citrus fruits were selected and periodically transferred through citrus fruits to maintain virulence. All of the plant pathogenic strains were cultured at 25°C on PDA plates for 1 week and stored at 4°C. Fruits Interdonato lemons (Citrus lemon) were obtained from local orchards in Antalya, Turkey and selected by hand. Fruit had not received any pre-harvest fungicide treatment. Selected lemons were sorted to remove any with apparent injuries or infections and were randomly assigned to different treatments and stored at 4°C for 3–5 d until used. Panomycocin preparation Panomycocin was prepared as described previously (Izgu and Altinbay 2004). Briefly P. anomala NCYC


434 cells were cultured to stationary phase in 1 L of YEPD at pH 4.5, at 20°C and at 120 rpm on a gyratory shaker (Innova 4330, New Brunswick Scientific, USA). Cell-free culture liquid was concentrated with 30 kDa-cutoff ultra-filter (Vivaspin, Sartorius AG, Goettingen, Germany). The crude protein was purified by ion-exchange chromatography with POROS HQ/M column (Perseptive Biosystems, Framinghan, MA) followed by gel filtration with a TSK 2000SW column (Tosoh, Tokyo, Japan). Purification steps were done on a fully automated HPLC system BioCAD 700E (Perseptive Biosystems, Framinghan, MA) which included an Advantec model SF120 fraction collector (Advantec MFS, Japan) Panomycocin concentration was estimated according to the method of Bradford (1976) using bovine serum albumin as a standard. The purity of Panomycocin was ensured by silver staining after linear gradient (5–20%) discontinuous SDS-PAGE. It was stored at -20°C in 0.1 mol l-1 Na2HPO4-citric acid buffer at pH 4.5, containing 20% (v/v) glycerol until used. In vitro susceptibility testing of Panomycocin The in vitro antifungal activity of Panomycocin was examined by the broth microdilution method described in document M38-A of the Clinical and Laboratory Standards Institute (CLSI 2002) with some adaptations. The growth medium used for broth microdilution susceptibility testing was RPMI 1640 medium without NaHCO3 but with L-glutamine (Cat no: R-6504, Sigma, St Louis) buffered to pH 4.5 with 0.1 M Na2HPO4-citric acid buffer. For the preparation of inocula composed of spores, the fungal isolates of P. digitatum DSMZ 2776, P. italicum DSMZ 2756, and the field isolates of P. digitatum (PHD-7 and PHD-9) and P. italicum (PHI-2 and PHI15) were subcultured on PDA plates at 25°C. After incubation for 7 d, cultures were flooded with sterile distilled water containing 0.05% (v/v) Tween 80 (Merck). Spores were gently scraped from fungal colonies with a sterile bacterial loop and transferred into a sterile tube. After heavy particles were allowed to settle for 5–10 min, the upper homogenous suspension was transferred, then filtered through a nylon membrane with a pore dimension of 11 lm (Millipore NY 1104700, USA). The resulting suspension was mixed by vortexing for 15 s. The

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concentration of the spore suspension was adjusted to 50 9 105 spores/ml by microscopic enumeration with a cell-counting haemocytometer. This suspension was diluted 1:50 in twofold concentrated RPMI 1640 to obtain an inoculum of approximately 105 spores ml-1. The minimum inhibitory concentrations (MICs) of Panomycocin were determined in 96-well flat-bottom microtitre plates. Each microdilution well containing 100 ll Panomycocin solution which was prepared in twofold dilution series ranging from 32 to 0.5 lg ml-1 in 0.1 mol l-1 Na2HPO4-citric acid buffer, pH 4.5 was inoculated with 100 ll of the spore suspension. This step diluted the medium (1X RPMI 1640), Panomycocin concentrations (16–0.25 lg ml-1) and inoculum densities (5 9 104 spores/ml) to the desired final concentrations. For each test plate, a Panomycocin-free growth control with growth medium containing an equivalent amount of Na2HPO4-citric acid buffer, pH 4.5 was included together with the sterility control. The microdilution plates were incubated at 25°C for 2 days. The MIC endpoints were determined both spectrophotometrically by measuring the absorbance at 492 nm using a microtitre plate reader (Spectramax 190, Molecular Devices, USA) at 12 h intervals for 48 h and visually with the aid of magnifying mirror by comparing the growth inhibition in each well with that of the growth control well. Panomycocin MICs were defined as follows: MIC-0 corresponds to the lowest Panomycocin concentration producing a clear well or 100% growth inhibition and MIC-2, the lowest Panomycocin concentration producing prominent growth reduction or a 50% reduction in growth. Each treatment was carried out in triplicate (three wells in the plate) and the mean and standard deviation (SD) (after background subtractions) were calculated for each. The experiments were replicated at least twice for each fungal strain. Fungal growth in each well of microtitre plates containing different concentrations of Panomycocin was also routinely checked every 6 h by an inverted microscope (Nicon, Melville, NY). Fruit decay test In vivo antifungal activity of Panomycocin was determined according to Lo´pez-Garcıá et al. (2003) with slight modifications. Experiments were carried out on freshly harvested lemon fruits which were not

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Results

(a)

(b)100 80

Inhibition %

treated with any antifungal agent. Fruits were surface sterilized by submerging in 5% commercial bleach solution for 5 min and subsequently washing 3 times with distilled water. After air drying, the fruits were treated with 70% ethanol. Fruits were again air dried and wounded (approximately 3 mm in depth) by making punctures at three sides around the equator of the fruit with the edge of a sterile micropipette tip (1000 ll). Fungal strains used in these experiments were P. digitatum DSMZ 2776 and PHD-7 and P. italicum DSMZ 2756 and PHI-15. The inocula contained 105/ml spores and 16 lg ml-1 Panomycocin were applied onto each wound. For the Panomycocin free growth control, spore suspensions mixed with 0.1 M Na2HPO4-citric acid buffer, pH 4.5. Fruits were incubated at 25°C and 90% relative humidity (RH) for 7 days. Each treatment was repeated three times with three fruits per replication.


60 40

P.italicum P.digitatum

20 0 0,25

0,5

1

2

4

8

16

Panomycocin concentration (µg /ml)

Fig. 1 a and b Inhibitory activity of Panomycocin on the growth of Penicillium spp. a (1) P. italicum DSMZ 2756 (2) P. digitatum DSMZ 2776. (A) Sterility control (B) Growth control. Wells a to g contain Panomycocin in twofold dilution series ranging from 0.25 to 16 lg ml-1

In vitro activity of Panomycocin The in vitro antifungal activity of Panomycocin was determined against P. digitatum and P. italicum as described in the methods. All the fungal isolates grew well on the culture media without any observable differences in their mycelial mass and they all produced adequate growth in microtiter plates within 2 days. Different standard concentrations of Panomycocin were tested in the experiment to better define the most active concentration. Figure 1 shows the effect of different concentrations of Panomycocin on the growth of P. digitatum DSMZ 2776 and P. italicum DSMZ 2756. All tested concentrations of Panomycocin reduced spore germination when compared with growth control. Panomycocin caused 50% reduction in the growth of P. digitatum and P. italicum isolates at concentrations of 2 and 1 lg ml-1, respectively. Complete inhibition (MIC0) of all the tested Penicillium spp. by Panomycocin were observed at a concentration of 16 lg ml-1. With the tested pathogens, growth inhibition increased with increased concentrations of Panomycocin. In vitro fungal growth inhibition mediated by Panomycocin was also analyzed microscopically. Growth of the tested strains in the wells containing

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different concentrations of Panomycocin was compared with the growth control well. After 18 h of incubation, complete inhibition of spore germination of the tested strains was observed at a Panomycocin concentration of 16 lg ml-1 which corresponds to MIC-0 value. At concentrations lower than the MIC0 value, significant growth anomalies like reduction in germination and germ tube elongation and mycelial growth were also observed. Panomycocin at 4 lg ml-1 markedly retarded the elongation of germ tubes and caused cell swelling (Fig. 2). The effect of Panomycocin on mycelial growth of phytopathogenic strains 48 h after starting the culture also visualized microscopically. At 4 lg ml-1 of Panomycocin, condensed hyphal aggregates were observed around the germinating spores compared with thin, elongated, well extended mycelial growth in Panomycocin free wells (Fig. 3). Inhibition of fungal infection of citrus fruit Laboratory controlled fruit inoculations were conducted to determine whether the observed in vitro antifungal properties of Panomycocin were in agreement with an inhibition of green and blue mold diseases of citrus fruit. We used a bioassay in which


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surface of the lesions and all of the control fruits were infected. At the end of fourth day, expanded lesions of blue and green mold diseases with blue and green spore masses formed on the control group. In contrast, no signs of green and blue mold diseases were detected on lemon fruits treated with Panomycocin. Panomycocin showed 100% protection of the lemon fruit against P. digitatum and P. italicum even after 5–7 days.

Discussion

Fig. 2 The effect of Panomycocin on germ tube elongation of P. digitatum DSMZ 2776 after 24 h incubation. a growth control well and b well containing 4 lg ml-1 of Panomycocin. Bars 40 lm

Fig. 3 The effect of Panomycocin on mycelial growth of P. italicum DSMZ 2756 after 48 h incubation. a growth control well and b well containing 4 lg ml-1 of Panomycocin. Bars 40 lm

spore suspensions of the both standard strains and field isolates (in the presence or absence of Panomycocin) were inoculated onto the wounded outer peel of lemons. Fig. 4 shows fungicidal activity of Panomycocin upon artificial infection of green mold on lemon fruit after 7 days. Under our conditions and inoculum concentration, at the end of second day, typical symptoms of the blue and green mold diseases, such as discolored watery spots and white mycelial network were detected in the control group. By the day three, white powdery growth of mycelium developed on the

Antifungal proteins have drawn attention for their potential application in plant disease control (Lo´pezGarcia et al. 2000). Due to both ecological concerns and personal health concern knowledge of the exact mode of action of an antifungal protein is a prerequisite for its application. Among the antifungal proteins with known mechanism of action, glucanases are under investigation as a promising group in providing protection against diseases. (Bar-Shimon et al. 2004; Ferrer 2006; Peng et al. 2009). In this work, we have demonstrated the potent growth inhibitory activity of Panomycocin, a novel exo-beta 1,3 glucanase, against isolates of P. digitatum and P. italicum that are the most important causes of post harvest decay in citrus fruits worldwide. Data obtained from the present study showed that Panomycocin exerted various levels of antifungal activity toward the tested organisms. In the presence of Panomycocin at concentrations less than MIC-0 value, abnormal morphological changes in fungal hyphae and spores were observed in the tested Penicillium spp. Our controlled in vivo investigations also demonstrated that treating pathogen-inoculated lemon fruit with Panomycocin at concentrations equal to their in vitro MIC-0 value significantly reduced the decay of lemon fruit and resulted in extended shelf life. These results underscore the potential for the application of Panomycocin as a method of plant protection. It is well known that surface pH of the injured or wounded citrus fruit decreases (the natural pH of lemon within 2 mm deep is around 5) and further decreases with age (Smilanick et al. 2005). Changes of the pH value of citrus fruit dramatically alters the effectiveness of chemical fungicides as neutral forms of chemical fungicides penetrate membranes of

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Fig. 4 Fungicidal activity of Panomycocin upon artificial infection of green mold on lemon fruit for 7 days after treatment. a inoculum contained 105/ ml spores of P. digitatum DSMZ 2776 and 16 lg ml-1 Panomycocin and b spore suspension mixed with 0.1 M Na2HPO4-citric acid buffer, pH 4.5

pathogens and are more toxic than charged forms (Lo´pez-Garcıá et al. 2003). Siegel et al. (1977) showed that imizalil (IMZ) was more toxic to P. italicum at pH 7 than at pH 5, and observed that little IMZ entered the mycelium at pH 5, compared with pH 7. It is noteworthy in this regard that Panomycocin retains its biological activity and provides protection towards pathogens under acidic conditions (pH 3–5.5). Lemon has become one of the most popular citrus fruit in the world and is cultivated in warmer climates across the globe. Though most people peel the lemon and use only the fruit part, lemon peel, on its own has many traditional uses and getting more popular in medicine due to its antioxidant properties (Bocco et al. 1998). Also essential oils recovered from citrus peels represent an increasing economic importance in the citrus by-product industry (Li et al. 2005). Accumulation of the residues of currently used chemical fungicides occurs principally in the peel, so the development of non toxic safer alternatives against phytopathogenic fungi for the control of post harvest decay becomes more important for citrus fruit as removal of chemical fungicides from the peel is very difficult by simple washing. In conclusion, Panomycocin is an attractive candidate for future antifungal compound in fresh citrus protection. Acknowledgments This work was supported by a grant from the Scientific and Technological Research Council of Turkey (TUBITAK) (Project no: 104T495).

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